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Doctoral Degrees (Medicine)

Permanent URI for this collectionhttps://hdl.handle.net/10413/8104

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Browsing Doctoral Degrees (Medicine) by SDG "SDG3"

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    Effect of HIV-1 subtype C Transactivator of transcription (Tat) A21P variant on TAR binding ability, nuclear levels of active positive transcription elongation factor b (P-TEFb) and viral latency.
    (2023) Mkhize, Zakithi Zinhle.; Madlala, Paradise Zamokuhle.
    The HIV-1 Transactivator of transcription (Tat) enhances the ability of the viral promoter 5’ long terminal repeat (LTR) to drive viral gene transcription and is important for HIV-1 pathogenesis. Tat binds to the transactivator RNA (TAR) element of the 5’LTR and subsequently recruits the host positive transcription elongation factor b (P-TEFb) for efficient viral gene transcription. Inter- and intra-subtype Tat genetic variation that translates to functional differences has been reported. Specifically, HIV-1 subtype C (HIV-1C) exhibiting Alanine at position 21 of the Tat protein (TatA21) was reported to be associated with reduced LTR transcriptional activity compared to Tat exhibiting Proline at position 21 mutation (TatP21). However, the effect of Tat variation on its ability to recruit P-TEFb is unknown. Therefore, this study seek to determine the effect of HIV-1 subtype C TatA21 mutant on the ability of Tat to recruit P-TEFb to 5’ LTR to enhance viral gene transcription. To this effect, site-directed mutagenesis (SDM) was performed on the Plasmid pcDNA3.1(+) HIV-1C BL43/02 TatA21 to introduce TatP21 alone or together with other mutations using designed primers and the Q5 DNA polymerase kit. The effect of Tat mutations was measured using Tat transactivation assay where the luciferase activity was the measured output in TZM-bl cell lines and the impact of TatA21 was further assessed on ability of the LTR to drive GFP and Gag expression in Jurkat and A72 cells respectively. Next, protein modelling was performed using Hdock software, followed by RNA immunoprecipitation (RNA IP) was performed using stably expressing TatA21 and TatP21 in Jurkat cells. Lastly, co-immunoprecipitation of TatA21 and associated with significantly reduced LTR transcription activity compared to TatP21 (p = 0.0004). TatA21 resulted in had significantly lower GFP expression Jurkat cells (p = 0.0439) and lower Gag expression in A72 cells compared to TatP21. Although TatA21 reduced the LTR transcription activity compared to TatP21, protein modelling using Hdock software revealed that TatA21 and TatP21 protein structures were the same. Consistently, molecular docking showed that TatA21 had a lower binding affinity than TatP21. The RNA IP showed that TatA21 had significantly reduced affinity to bind to TAR compared to TatP21 (p = 0.0151). Moreover, TatA21 and TatP21 formed a complex with cycT1 and CDK9. Taken together, our data shows that HIV-1C TatA21 significantly reduced its transactivation activity but does not affect its ability to recruit P-TEFb. Interestingly, TatP21 is able to bind TAR more efficiently than TatA21 thus revealing a possible mechanism but which the reduced functionality of SDMs and patient derived TatA21 variants was observed. The effect of TatA21 and TatP21 on the propensity of HIV-1 latency development or reversal. To this effect, a recombinant viral vector exhibiting either TatA21 (C731CTatA21C) or TatP21 (C731CTatP21C) were generated. The C731CTatA21C or C731CTatP21C were separately co-transfected together with VSV-G and R8.91 into Jurkat cells for virus production. This virus was then used to infect Jurkat cells for 3 days. Followed by cell sorting of GFP- cells, which represented either truly negative or latently infected cells was then performed. We were able to successfully generate C731CTatA21C virus and characterized it to a 1.2% reactivation. However, the generation of C731CTatP21C recombinant viral vector was unsuccessful and thus could not be used for comparison. Future studies should involve the characterization of TatP21 in the propensity of latency development and/ or reactivation.
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    Immune biomarkers of pulmonary tuberculosis treatment response and disease severity among HIV-infected and uninfected individuals from Kwazulu-Natal, South Africa.
    (2023) Rambaran, Santhuri.; Sivro, Aida.; Naidoo, Kogieleum.
    Background: Tuberculosis is one of the major causes of morbidity and mortality worldwide. The COVID -19 pandemic has had a devastating impact on TB, contributing to increased incidence of both TB and drug-resistant TB. Identification of host immune biomarkers of TB risk, treatment outcome and disease severity are key to the development of more efficient diagnostics and treatment modalities. There is an urgent need for accurate and easily detectable non-sputum-based biomarkers that can correlate with the activity or burden of Mycobacterium tuberculosis. Here, we characterised soluble and cellular phenotypes during active TB and TB/HIV co-infection and assessed their associations with time to negative culture conversion and disease severity. Methods: The study was performed utilizing stored plasma and peripheral blood mononuclear cells from the Improving Retreatment Success (IMPRESS) trial. Multiplex immunoassays and ELISAs were used to evaluate 24 cytokine and chemokine expression during active TB (n=132). Flow cytometry was used to evaluate phenotypic profiles of monocytes, dendritic cells (n=90) and CD4+ T cells (n=75). A Cox proportional hazards and logistic regression models were used to assess the associations between the measured cytokines and chemokines, phenotypic profiles of monocytes, dendritic cells and CD4+ T cells and time to negative culture conversion and lung cavitation in individuals with TB and TB/HIV co-infection. Results: We identified soluble inflammatory signatures of treatment response and disease severity. IP-10 expression during active TB was associated with increased odds of sputum culture conversion by 8-weeks in the total cohort and among the HIV-infected individuals. Increased MCP-3 expression was associated with a shorter time to culture conversion in the total cohort. While among the HIV-infected individuals, higher expression of IL-1RA, IP-10 and IL-1α associated with a shorter time to culture conversion. Higher expression of IL-6 was significantly associated with shorter time to culture conversion and increased risk of lung cavitation in the overall cohort and among TB/HIV co-infected individuals. Additionally, higher IL-1RA expression was associated with the presence of lung cavitation in the total cohort and in HIV-infected individuals. We observed distinct monocyte and dendritic cell profiles in TB/HIV co-infection. Individuals with TB/HIV co-infection had a significantly higher percentage of total monocytes and dendritic cells compared to healthy controls. Increase in CCR2, CD11b and CD40 was associated with active TB while decrease in CX3CR1 and increase in CD163 was associated with HIV infection. Expression of CX3CR1 on non-classical monocytes was associated with longer time to culture conversion while expression of CD86 on intermediate monocytes was associated with presence of lung cavitation. With respect to CD4+ T cells HIV positive individuals with active TB had significantly lower percentage of CD4+ T cells and significantly higher proportion of activated CD4+ T cells compared to TB and healthy control groups. Percentage of CD4+ T cells was significantly associated with increased risk, while the percentage of activated CD4+ T cells was associated with decreased risk of lung cavitation. Integrin α4β7 expressing CD4+ T cells were increased in TB/HIV compared to TB group and was associated with longer time to TB culture conversion in co-infected individuals. Conclusion: The data from this study provides valuable insight into the role that plasma immune biomarkers, monocytes, dendritic and CD4+ T cells play in TB treatment response and disease severity in active TB and TB/HIV co-infection.